Proximity-driven site-specific cyclization of phage-displayed peptides

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3.


Materials:
Unless otherwise stated, all materials and chemicals were purchased from Sigma-Aldrich, Invitrogen or Thermo Fisher Scientific and used with no further adjustment or purification.All restriction/ protease enzymes and ligation/ polymerase chain reaction (PCR)/ site-directed mutagenesis (SDM) master mixes were purchased from New England Biolabs (NEB).All primers and oligonucleotides were obtained from Sigma-Aldrich and string inserts from GeneArt (Thermo Fisher Scientific).
The following materials were prepared by AstraZeneca: 2xTYAG petri and bioassay plates, relevant antibiotics (ampicillin, kanamycin, chloramphenicol), 20% (v/v) glucose solution, 50% (v/v) glycerol solution and materials for protein expression and purification, Kunkel mutagenesis, phage production and precipitation, and phage display selections.
The plasmid maps and full-length protein sequences associated with AstraZeneca cannot be shared or discussed due to proprietary reasons.

Supplier Genotype
Mix and go! Dh5-α Zymo The synthesis of CPO-PFP and CPO-PEG2-biotin were achieved following a protocol previously reported by our group. 1i.
binding responses, represented as changes in interference (nm), are shown as a function of time (seconds). != Calculation of the binding affinity constant (Kd) of peptide Str1-CCA (analysis performed using Biacore 8K Evaluation Software).The data corresponds to one of three independent replicates.Experiments were performed in triplicate.